Receptors for estrogens and progesterone in the porcine cervix

In vitro binding and exchange methods were used to determine the levels of estradiol and progesterone receptors in cytosolic and nuclear fractions of cells obtained from the porcine cervix at different stages of the estrous cycle. The concentration of estradiol cytosolic receptors was about 4500 sites/cell during the luteal phase and increased to a maximum of approximately 7600 sites/cell on day 1 of the cycle, decreasing to a level of 2700 sites/cell on days 3-4. The estradiol nuclear receptor level increased between the end of the luteal phase and the onset of heat from 300 to 1200 sites/cell. No reduction in the number of nuclear sites was seen between day 1 and 3-4. The level of the progesterone cytosolic receptor and its cycle profile was very similar to that of the estradiol receptor. The nuclear receptor, however, reached its lowest level of 760 sites/cell on day 1 of the cycle, increased to a value of 4700 sites on days 3-4 and showed a steady level of about 1000 sites/cell during the luteal phase. The data obtained agree with present theories on the endocrine mechanisms regulating receptor levels in the uterus. Furthermore, these data support a concept in which the constriction of the cervix occurring in response to increased concentrations of circulating estradiol is mediated via steroid receptors.     

Effect of estradiol and progesterone on oviductal LH-receptors and LH-dependent relaxation of the porcine oviduct

We have previously shown that the porcine oviduct possesses immunoreactive and functional LH receptors and that LH causes relaxation of the oviduct, especially during the periovulatory stage of estrous cycle. The current studies were undertaken to investigate the effects of estradiol and progesterone on LH receptor protein and LH-stimulated motility of the oviduct in steroid-primed ovariectomized gilts. Twenty-one cross-bred gilts were ovariectomized at 6 m.o. of age. Four weeks later gilts received daily intramuscular injection of either 2 mL corn oil (control n = 4), estradiol benzoate (EB) 1.5 mg (n = 6), progesterone 50 mg (n = 5), or 1.5 mg EB plus 50 mg progesterone (n = 6) for 4 consecutive days. The gilts were slaughtered on Day 5 after the first injection of steroids or vehicle. Rings of isthmus and ampulla were collected from each oviduct and placed in a tissue chamber perfused with Kreb's solution for 60 min. The mechanical activity was recorded for 30 min after LH treatment. Immunoreactivity of LHR in the Fallopian tube sections were detected in the epithelium of the tubal mucosa, smooth muscle cells and the blood vessel endothelium. Western blotting showed that porcine oviducts contain 75, 48 and 45 kDa immunoreactive LH receptor proteins, like the corpus luteum (CL). The lowest receptor expression was found in controls and in gilts treated with estradiol or progesterone. Combined treatment with estradiol and progesterone resulted in a significant increase of LH receptor protein concentrations when compared with control animals. In vitro LH treatment affected oviduct contractility of combined estradiol and progesterone treated gilts but not the oviduct of the remaining groups. It also caused a decrease in amplitude, frequency and areas under the curve (AUC) of ampulla (P < 0.05) and the amplitude and AUC of isthmus (P < 0.001) in combined estradiol and progesterone-primed gilts. These results indicate that estradiol and progesterone together, but not separately, increase LH receptor protein in the porcine oviduct and that combined estradiol and progesterone priming is necessary for LH-induced relaxation of the porcine oviduct.     

Biosynthesis and posttranslational finishing of the estradiol receptor

The time course of subcellular receptor distribution in porcine endometrial epithelium was studied after intrauterine administration of estradiol alone or in combination with puromycin. In untreated cells, the major proportion of receptor is associated with cytoplasmic membranes. The solubilization of receptor from isolated nuclei is independent of their estradiol content. Smooth cytoplasmic membranes are the site of origin of receptor which is swiftly translocated into the nucleus in a 1:1 ratio with the hormone after exposure of the cells to estradiol. Simultaneously administered puromycin delays receptor synthesis and reveals that the nuclear passage of receptor is terminated by receptor degradation. The synthesis of receptor proceeds in rough endoplasmic membranes. A subsequent finishing and deposition in smooth membranes depends on intact protein synthesis.     

Significance of lipoidal estradiol in human mammary tumors

Incubations of p-nitrophenyl fatty acyl esters and estradiol-17 beta fatty acyl 17-esters with porcine esterase, human mammary tumor cytosol and rat uterine cytosol leads to ester hydrolysis of compounds with short chain fatty acids. Esters with long chain fatty acids show no hydrolysis except in the presence of Tween 80. Short chain fatty acid esters have a higher binding potency to the estrogen receptor than long chain fatty acid esters. Extraction of the nuclear receptor peak sedimenting at 4.6S and identification of the steroid showed that about 90% of the radioactivity was associated with estradiol and only 10% with estradiol esters. These studies show that estradiol fatty acyl esters act as a storage form from which estradiol is released by enzymatic hydrolysis.     

Effects of estradiol concentration on levels of nuclear estrogen receptors in MCF-7 breast tumor cells

We have measured the time-course of estrogen receptor levels in nuclei of the estrogen-responsive breast tumor cell line MCF-7 during 90-120 min exposure of the cells to estradiol at physiologic (10(-10)M), pharmacologic (10(-6)M), and an intermediate (10(-8)M) concentration. Cells were preincubated for one week in a serum-free defined medium resembling that of Barnes and Sato, and then incubated in estradiol-containing medium. Nuclei were isolated at various times during the incubation, and filled and unfilled nuclear estrogen receptor levels were assayed. Increasing the concentration of estradiol in the incubation medium from 10(-10)M to 10(-8)M yielded increasing levels of filled nuclear receptor at all times studied, while further increase of the estradiol concentration of 10(-6)M decreased filled receptor levels from 10(-8)M values. Unfilled receptor levels dropped rapidly to zero under 10(-6)M and 10(-8)M estradiol incubation, but remained unchanged under 10(-10)M estradiol incubation. Together these results suggest that high-concentration estradiol may lead to "down-regulation" of filled nuclear receptors, which may be a contributing factor in inhibition of tumor growth. On the other hand, the continued presence of unfilled receptors only under physiological concentrations of estradiol may suggest a role for these receptors in sustaining tumor growth.     

The characteristic binding of catechol estrogens to estrogen receptors in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

The binding of catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol) to estrogen receptors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor cytosols was investigated. Cytosol estrogen receptors exhibited high affinities (Ka = 1.12-1.88 X 10(8) M-1) for all catechol estrogens as well as estradiol. The receptor level of catechol estrogens (46.1-97.5 fmol/mg protein) was 1.6-3.0 times higher than that of estradiol; especially the binding of 4-hydroxyestrone to estrogen receptors was the highest of all catechol estrogens and estradiol. In judging the receptor level of more than 20 fmol/mg protein to be positive, the binding of catechol estrogens to estrogen receptors was approximately correlated with that of estradiol. The positive receptor level of catechol estrogens was found in a half of tumor cytosols which showed the negative receptor level of estradiol. These results suggested that characteristic estrogen receptors indicating high affinities for catechol estrogens might be present in rat mammary tumor cytosols.     

Characterization of platelet-activating factor receptors in porcine platelets

Despite a large number of studies describing the properties and effects of platelet-activating factor (PAF), little is known about its receptor structure. The characterization of the PAF receptor from additional cell types and species is important for the design of strategies to purify and characterize the receptor molecule. Porcine platelets were shown to bind PAF with characteristics similar to several other species, based on receptor number, affinity, and the activity of PAF antagonists. We found that the affinity for binding was higher in porcine than in rabbit platelets (Kd = 0.68 +/- 0.13 nM for rabbit and 0.29 +/- 0.10 nM for porcine). Porcine platelets have approximately 281 +/- 158 receptors per cell compared with 689 +/- 229 receptors in rabbit platelets. Rabbit platelets respond to concentrations of PAF that are approximately 10(5)-fold lower than those required for aggregation of porcine platelets, but this difference is probably not due to the differences in receptor number alone. When binding was compared between purified membranes from these two cell types, porcine platelets had 20-fold fewer receptors per milligram of membrane protein, but this difference may have been due to an artifact of the membrane preparation procedure. Binding of PAF was severely hindered at cold temperatures. It was undetectable in whole cells on ice and greatly reduced with purified membranes. This study is the first to characterize PAF receptors in porcine platelets, which represent a potentially useful source of receptor for further biochemical characterization.     

Discrete early changes in cellular subpopulations of rat uterine and anterior pituitary estrogen receptors in response to acute exposure to exogenous estradiol

Distribution of estrogen receptors among ligand-occupied and unoccupied species in cytosolic and nuclear subcellular compartments has been analyzed as an acute response to administration of 5 micrograms of estradiol in adult female rats. Patterns of anterior pituitary and uterine receptor turnover were monitored at intervals over a 5-h period, using either intact or 2-weeks ovariectomized animals. In terms of total cellular receptor content, initial levels were higher in castrate animals, but rapidly fell to intact levels within an hour following estradiol injection. Cycloheximide given shortly before estradiol had no effect on total pituitary receptor patterns, but appeared to result in an elevation in total uterine receptor content at early intervals. Unoccupied cytosol receptors were rapidly depleted and, with the exception of castrate pituitary samples, showed some replenishment within 5 h, all of which was cycloheximide-sensitive. Initially, occupied cytosol receptors were low in intact rats, but were present at levels approaching those of the unoccupied cytosol receptor forms in the ovariectomized rat tissues. Occupied cytosol receptor levels fluctuated in response to estradiol. Subpopulations of nuclear receptors, especially the unoccupied species, showed significant tissue specificity. In the uterus, unoccupied nuclear forms were initially present in high amounts, and the levels did not change in response to estradiol administration. In the pituitary, the levels of these receptors rose and subsequently fell over the 5-h interval. Cycloheximide conferred a similar biphasic response to estradiol upon the otherwise insensitive unoccupied nuclear forms of the uterus. Occupied nuclear receptors turned over completely during the 5-h study interval, with the kinetics being faster in the castrate than the intact tissues. Cycloheximide affected occupied nuclear forms of the uterus only, dramatically increasing their levels in response to estrogen and causing prolonged retention in the castrate animal model. Collectively, the cycloheximide effects on this system are consistent with early estrogen induction or stimulation of a protein which inhibits accumulation of occupied or unoccupied receptor species within the nucleus. This re-examination of all forms of cellular estrogen receptors as they fluctuate acutely in response to exogenous estrogen has revealed several heretofore undetected responses which must be incorporated into the overall scheme of early estrogen action.     

Hormonal control of implantation in guinea pigs

In the guinea pig, for which implantation is supposedly progesterone-dependent, actual hormonal requirements were assessed by measuring the levels of circulating estradiol and progesterone and correlating them with their content in the ovaries and uterus, and uterine concentrations of their receptors prior to, during, and immediately after implantation. Ovarian and uterine content and plasma levels of estradiol and progesterone, as well as uterine cytosolic receptors of these two hormones, were high at proestrus. Up to day 3 of pregnancy, estradiol remained high in peripheral plasma, ovarian and uterine tissues, but reached low levels at the time of implantation. The levels of progesterone showed a gradual increase in plasma and ovaries till the time of implantation, with the embryonic site of the uterus accumulating more of progesterone compared to estradiol. As pregnancy progressed, a gradual translocation of cytosolic to nuclear receptors occurred, both with estradiol and progesterone receptors. Comparing the receptor values for estradiol at each uterine site showed no significant alterations between embryonic and interembryonic cytosolic receptors. While significantly high levels of nuclear estradiol receptor were found at the inter-embryonic site on day 9 of pregnancy, the cytosolic and nuclear progesterone receptor concentrations were greater at the embryonic site on the same day. These findings demonstrated that the uterus is adequately exposed to estradiol and progesterone prior to ovulation and again in early pregnancy (day 1-3), thus facilitating implantation in the guinea pig (on days 7-8).     

Effect of cyanoketone on follicle-stimulating hormone (FSH) induction of receptors for FSH in granulosa cells of the rat

Follicle-stimulating hormone (FSH) enhances the conversion of testosterone or androstenedione into estradiol by stimulating the aromatase enzyme system. Estradiol also enhances FSH action. Thus, a synergistic action of FSH and estradiol may be required for maturation of ovarian follicles. We hypothesized that estradiol may be required for FSH action. Thus, blocking estrogen synthesis should prevent FSH-induced increases in FSH receptors. Hypophysectomized rats were divided into five groups and injected subcutaneously with: 1) saline, 2) cyanoketone (0.05 mg, blocks the conversion of pregnenolone to progesterone), 3) ovine FSH (oFSH, 200 micrograms), 4) cyanoketone then oFSH 24 h later, or 5) cyanoketone plus estradiol [or progesterone, testosterone, promegestrone (R5020), dihydrotestosterone (DHT), 2 mg], then FSH 24 h later. Animals were decapitated at 0, 12 or 24 h after an injection of oFSH, and membrane receptors for FSH and luteinizing hormone (LH), plus nuclear receptors for estradiol from granulosa cells, were measured. LH receptor levels were increased only after administration of FSH and estradiol. At 0 and 24 h, numbers of FSH or estradiol receptors were similar in saline- and cyanoketone-treated animals. FSH alone increased (P less than 0.01) FSH and estradiol receptors 3-fold and 4-fold, respectively, over controls by 12 and 24 h. Cyanoketone prevented these increases in FSH and estradiol receptors. Estradiol replacement fully reversed the effects of cyanoketone on FSH action. Replacement with progesterone and testosterone was able to only partially restore levels of FSH receptors; however, estradiol receptor numbers were also increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.     

Localization of estrogen receptors in uterine cells. An appraisal of translocation.

The nuclear localization of estrogen receptors has been examined under conditions which minimize redistribution and localization artifacts. A procedure is presented which rapidly lyses suspensions of cells from immature rat uteri by using 0.04% Triton X-100 in isotonic buffer. The ‘nuclei’ which are obtained after lysis have a median diameter of 1μm and are devoid of nuclear membranes. There is close agreement between the number of cells before lysis and the number of nuclear particles after lysis. Triton X-100 gave no interference with quantitative binding of estradiol to receptor and no alteration in the sedimentation behavior of receptor on sucrose gradients containing high or low salt. Using this procedure to monitor the dynamics of estrogen receptor distribution within uterine cells after exposure to estradiol, translocation of estrogen receptor to the nucleus was observed to occur at a rate slightly slower than the rate at which estradiol was specifically bound to free cells or receptors. The difference in these rates is compatible with a model in which estradiol must first bind to the receptor before the binding complex moves to the nucleus. The rate of nuclear translocation was temperature-dependent and was observed to occur at 0 °C, provided that enough time was allowed for steroid entry, receptor charging and transit to the nucleus. Two distinct phases were observed to characterize nuclear receptor localization. In the first phase after hormone exposure, estrogen receptor progressively accumulated in the nucleus; afterwards, estrogen receptor was progressively lost from the nucleus but could not be detected in other subcellular compartments in a form still binding hormone. Since high cell viability was maintained during these manipulations, loss of nuclear receptor was not due to cell damage during in vitro incubation. These studies suggest that this decline in nuclear receptor level after hormone interaction, which is known to occur in vivo, may be a normal event during estrogen interaction with target cells.     

Alphafetoprotein is not a component of the 8 S estradiol receptor from the immature mouse uterus or brain.

The hypothesis that alphafetoprotein (AFP) could be a consitituent of the estradiol receptor in the uterus and brain of the immature mouse has been tested. Cytosols prepared from 2 week-old mice were depleted of AFP by immunoadsorption with a Sepharose-coupled anti-mouse AFP anti-serum. Aliquots of these cytosols containing nondetectable levels of AFP by RIA were then brought to 0.4 M KCl to convert 8 S estradiol receptors to the 4-5 S forms, reacted either with anti-AFP or with a control anti-IgG immunoadsorbent, labeled with 3H-estradiol, and centrifuged on glycerol gradients. There was no, or very little, loss of estradiol receptors in the experimental as compared to the control cytosols indicating that AFP is not a constituent of the 8 S estradiol receptor.     

Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing

The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.     

Antagonism of estrogen-induced prolactin release by progesterone

Previous work from our laboratory has shown that during the process of nuclear occupancy of the progesterone receptor complex (1-2 h), nuclear estradiol receptors of the anterior pituitary are depleted. The purpose of this study was to determine whether the depletion of nuclear estradiol receptors by progesterone had functional biological significance. The ovariectomized (26 days of age) immature rat was used as the model for analysis of this question. The ability of estradiol to release prolactin from the anterior pituitary was the function chosen to determine the biological significance of the progesterone and estradiol interactions. In response to estradiol exposure (2 micrograms/rat), prolactin release reached peak values from 8 h to 12 h and returned to control levels by 24 h. A second injection of estradiol 13 h after the initial injection stimulated a second increase in serum prolactin at 25 h. This model of two injections of estradiol 13 h apart served to provide adequate levels of anterior pituitary progesterone receptors and elevated serum prolactin levels upon which superimposed progestin modulation could be examined. A single injection of progesterone (0.8 mg/kg BW) 1 h before the second estradiol injection blocked the increase in serum prolactin. This action was a receptor-mediated event because progesterone had no effect without estrogen priming or when the progesterone antagonist RU486 was used. Finally, when the interval between the progesterone and second estradiol injection was extended to 4 h, a time period when progesterone does not deplete pituitary nuclear estrogen receptors, the estrogen-induced increase in serum prolactin was not blocked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct effect of estradiol on the number of dopaminergic receptors in the anterior pituitary, in ovariectomized rats

The effect of estradiol on anterior pituitary dopaminergic receptor content was studied in vivo and in vitro, in relation with the serum PRL secretion. A progressive and significant decrease in the number of these receptors was observed, a few hours before the serum release of PRL induced in ovariectomized females by a sequential treatment with different doses of estradiol benzoate. This decrease in the number of dopaminergic membrane receptors can be obtained as well in vitro, when anterior pituitaries, from ovariectomized rats, are incubated with 17 beta-estradiol. These results suggest that the stimulatory effect of estradiol on PRL secretion may be due, at least in part, to the direct "desensitization" to DA of anterior pituitary cells, which is produced by the decrease of dopaminergic receptor level.     

Characterization of the porcine ACTH receptor with the aid of a monoclonal antibody

Monoclonal antibodies were raised against the ACTH receptor by immunization of BALB1 c mice with porcine adrenal cortex cell membranes. Competition and binding experiments confirmed that one of these antibodies, IV/14/9, reacted with the ACTH receptor in competition with ACTH and caused a definite ACTH-like effect. This antibody was used to characterize the hormone receptor of the porcine adrenal cortex. The number of antibody binding sites per cell was calculated from a Scatchard plot to be 124,100 +/- 10,000. The curve was linear indicating the existence of a single class of receptors. The finding that a high concentration of IV/14/9 totally suppressed maximally ACTH-induced steroidogenesis confirms the view that only a single class of ACTH receptors exists. The antibody IV/14/9 neither reacted with intact porcine thymus cells nor with normal porcine lymphocytes but was bound to the mouse adrenal tumor cell line Y1 and to normal rat adrenal cortex cells with a low affinity. Two dimensional electrophoresis of lysates of porcine adrenal cortex cells and subsequent blotting of the proteins on nitrocellulose, using IV/14/9 as a primary and anti-mouse Ig as a secondary reagent, permitted the detection of three forms of the receptor, two of which had an identical apparent molecular mass of about 85,000 Da (isoelectric points: 6.14 and 6.27). The third was somewhat larger (94,000 Da and pI = 6.21).     

Purification of the alpha 2-adrenergic receptor from porcine brain using a yohimbine-agarose affinity matrix

A procedure has been developed for purification of the porcine brain alpha 2-adrenergic receptor to homogeneity. alpha 2-Adrenergic receptors were solubilized from porcine brain particulate preparations using sequential extraction into sodium cholate- and digitonin-containing buffers. The alpha 2-adrenergic receptors in the digitonin extract were identified using the alpha 2-adrenergic selective antagonist, [3H]yohimbine, and demonstrated the same specificity for interaction with adrenergic ligands as did the receptors in particulate preparations. Extraction into digitonin-containing buffers eliminated the modulation of receptor-agonist interactions by guanine nucleotides, but not by monovalent cations. A novel affinity resin, yohimbine-agarose, was synthesized and used for purification of alpha 2-adrenergic receptors. Using two sequential yohimbine-agarose affinity chromatography steps, digitonin-solubilized alpha 2-adrenergic receptors from porcine brain cortex were purified to homogeneity as assessed by radioiodination and silver stain analysis of these preparations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified alpha 2-adrenergic receptor has an approximate Mr = 65,000, as determined by photolabeling of the adrenergic ligand-binding subunit. The yohimbine-agarose affinity resin should be useful for purifying quantities of receptor sufficient for studies of receptor structure and function.     

Biological actions of monoclonal luteinizing hormone/human chorionic gonadotropin receptor antibodies.

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.